Anti-hiv dual specificity antibodies and methods of hiv treatment

ABSTRACT

Dual variable domain immunoglobulins (DVD Igs) are provided capable of tetravalent binding to bispecific sites of the human immunodeficiency virus (HIV). The DVD Igs may be asymmetric and may have more variable domains on either the light chain or the heavy chain of the Igs. The DVD Igs may have specificity for gp41 and gp120. Therapies are provided using DVD Igs to neutralize HIV viral loads.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application61/858,149 filed Jul. 25, 2013, which is incorporated herein byreference.

FIELD

The present invention relates generally to anti-HIV antibodies andantibody-like molecules and, in particular though non-limitingembodiments, to double-variable domain antibodies and methods oftreating an HIV infection.

BACKGROUND

The lentivirus human immunodeficiency virus (HIV) causes AcquiredImmunodeficiency Syndrome (AIDS), a condition in humans in whichprogressive failure of the immune system allows life-threateningopportunistic infections and cancers to thrive. HIV/AIDS is a globalpandemic, with most recent World Health Organization studies estimatingnearly 34 million people are infected with HIV. This figure includesover 3 million children under the age of 15. Currently, no cure for HIVexists.

The HIV virus infects a large number of different cells in the body,including various cell types of the immune system, but its infection ofCD4 T-lymphocytes largely underlies HIV pathogenesis. HIV-infectionleads to reduced CD4 T-lymphocytes, further leading to progressive lossof cell-mediated immunity and an increased susceptibility toopportunistic infections.

The HIV virus consists of a viral envelope enclosing a capsid, whichitself encloses the viral genome. The HIV envelope protein (Env)consists of precursor gp160 of the transmembrane domain gp41 (e.g., SEQID NO 1, which is one of many sequences for gp41) and the externaldomain gp120 (e.g. SEQ ID NO 2, which is one of many sequences forgp120), which are involved in virus-cell attachment. Mechanistically,gp120 attaches to the CD4 molecule present on T-lymphocytes, a series ofconformational changes occur with gp120 and gp41, and gp41 mediates thefusion of the viral and cellular membranes and insertion of viral coreand the genomic material into the target cell, resulting in host cellinfection.

Conventional neutralizing antibodies generally consist of two identicalheavy chains and two identical light chains, each with a single variabledomain (V_(H) or V_(L)) at the N-termini of the molecule. More recently,neutralizing antibodies have been adapted to include a second variableregion connected via a linker (L) sequence at the N-termini of thevariable domains of a conventional molecule and are generally referredto as a dual variable domain immunoglobulins (DVD-Igs). DVD-Igs areimmunoglobulin-derived molecules that contain two unique variabledomains (V domains) linked to a constant region with the capability oftetravalent, bispecific binding, while retaining affinity andspecificity of each of the parental antibodies. For example, DVD-Igshave been constructed that can bind both IL1a and IL1b, or IL-12 andIL-18. DVD-Igs have been proven effective in vitro and in vivo, andretain pharmacokinetic properties of the parental antibodies.

The idea of targeting two separate antigenic sites with a singleantibody has also been directed against HIV. The most common approachhas been to construct dual domain antibodies using an anti-gp120V-region fused to CD4. When the inter-domain linker length wasoptimized, enhanced neutralization by these CD4-anti-gp120immunoadhesins was obtained. Bi-specific antibodies with one V-domainagainst gp41 and one against gp120 have been produced; however theantibodies do not neutralize the virus as well as embodiments of thepresent invention. The failure to make effective neutralizing antibodiesis due in part to the enormous sequence diversity of HIV-1, and therelative inaccessibility of conserved domains of the HIV virus.

Accordingly, there is need for novel antibodies and antibody likemolecules and methods of neutralizing and eradicating HIV.

SUMMARY

In an exemplary embodiment of the present invention, an antibody isprovided, including: at least one variable domain with binding affinityto HIV gp120 and at least one variable domain with binding affinity toHIV gp41. At least one of a heavy chain and a light chain of theantibody has a variable domain with binding affinity to HIV gp120 linkedby a linker to a variable domain with binding affinity to HIV gp41. Incertain embodiments, only one of the heavy chain and the light chain mayhave two variable domains. In certain embodiments, both the heavy chainand light chain may have two variable domains.

The linker may be one of a helical linker and a flexible linker. Thelinker may be one of SEQ ID NO:9 and SEQ ID NO:10. The antibody mayinclude both chains of full-length 7B2 antibody. The heavy chain mayhave one variable domain with binding affinity to HIV gp41 and the lightchain may have a first variable domain with binding affinity to HIVgp120 linked by the linker to a second variable domain with bindingaffinity to HIV gp41. The light chain may have one variable domain withbinding affinity to gp41 and the heavy chain may have a first variabledomain with binding affinity to HIV gp120 linked by the linker to asecond variable domain with binding affinity to HIV gp41. Both the lightchain and the heavy chain may have a first variable domain with bindingaffinity to HIV gp120 linked by the linker to a second variable domainwith binding affinity to HIV gp41.

The heavy chain of the antibody may be one of SEQ ID NO 3, SEQ ID NO 4,and SEQ ID NO 8. The light chain of the antibody may be one of SEQ ID NO5, SEQ ID NO 6 and SEQ ID NO 7. The light chain is not SEQ ID NO 7 whenthe heavy chain is SEQ ID NO 8. The at least one variable domain withbinding affinity to HIV gp120 may include domains 1 and 2 of CD4.

In an exemplary embodiment of the present invention, a method oftreating an HIV infection is provided, including: administering to apatient infected with HIV at least one of an antibody and a geneticconstruct capable of producing the antibody in the patient, saidantibody having: at least one domain with binding affinity to HIV gp120;and at least one domain with binding affinity to HIV gp41. At least oneof a heavy chain and a light chain of the antibody has a domain withbinding affinity to HIV gp120 linked by a linker to a domain withbinding affinity to HIV gp41. The antibody may have been incorporatedinto an immunoconjugate having at least one of a toxin and a cytotoxicagent.

In an exemplary embodiment of the present invention, a method ofneutralizing HIV virus is provided, including: administering at leastone of an antibody and a genetic construct capable of producing theantibody, said antibody having: at least one domain with bindingaffinity to HIV 120; and at least one domain with binding affinity toHIV gp41. At least one of a heavy chain and a light chain of theantibody has a domain with binding affinity to HIV gp120 linked by alinker to a domain with binding affinity to HIV gp41. The least one ofan antibody and a genetic construct capable of producing the antibodymay be administered to a subject infected with HIV. The least one of anantibody and a genetic construct capable of producing the antibody maybe administered to a subject at risk of exposure to HIV.

DESCRIPTION OF DRAWINGS

FIG. 1 is a set of three graphs showing binding qualities of constructsand parental antibodies to antigens, according to an exemplaryembodiment of the present invention.

FIG. 2 is a set of two graphs showing indirect immunofluorescence andflow cytometry analysis of binding qualities to persistently infectedH9/NL4-3 cells, according to an exemplary embodiment of the presentinvention.

FIG. 3 is a set of two graphs showing indirect immonoconjugate assays ofanti-gp120 and anti-gp41/gp120 constructs, according to an exemplaryembodiment of the present invention.

FIG. 4 is a set of four graphs showing assay results for neutralizationof viral isolates, according to an exemplary embodiment of the presentinvention.

FIG. 5 is a set of three graphs showing assay results for neutralizationof viral isolates, according to an exemplary embodiment of the presentinvention.

FIG. 6 is a graph showing assay results for antibody-dependent cellularviral inhibition, according to an exemplary embodiment of the presentinvention.

FIG. 7 is a graph showing assay results for antibody-dependentphagocytosis, according to an exemplary embodiment of the presentinvention.

DETAILED DESCRIPTION

The present invention relates generally to bispecific antibodies,asymmetric antibodies, antibody-like molecules and methods of treatingHIV infections. Before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notnecessarily limited in its application to the details set forth in thefollowing description or exemplified by any examples. The invention iscapable of other embodiments or of being practiced or carried out invarious ways.

Throughout this disclosure, the term “antibody” may indicate a classicalantibody, but also an antibody-like molecule, a protein, a fragmentthereof, or any combination of these. “Antibody” may include proteins,fragments, glycoproteins (e.g., CD4), or portions thereof attached orlinked to antibodies and/or variable domains of antibodies. Sequencesencoding CD4 (domains 1 and 2) are referred to as variable domains inthis application. These terms are intended to be illustrative in nature,and certainly not limiting.

Any antibody or antibody fragment of the present invention, whetherattached to other sequences or not, can also include insertions,deletions, substitutions, or other selected modification or particularregions or specific amino acids residues, provided the activity of theantibody or antibody fragment is not significantly altered or impairedcompared to the non-modified antibody or antibody fragment. Thesemodifications can provide for some additional property.

Embodiments of the present invention may be administered to a subject ina pharmaceutical composition, which may be any acceptable carrier.Effective dosages and schedules for administering embodiments of thepresent invention may be determined empirically. Embodiments may beadministered to neutralize, treat, prevent or eradicate HIV infection.Embodiments of the present invention may be used in gene therapytechniques for HIV. In certain embodiments, genetic constructs capableof inducing production of antibodies of the present invention may beadministered to a patient in need thereof.

Embodiments of the present invention provide an antibody having at leastone variable domain with binding affinity to HIV gp120 and at least onevariable domain with binding affinity to HIV gp41. At least one of aheavy chain and a light chain of the antibody may have a variable domainwith binding affinity to HIV gp120 linked by a linker to a variabledomain with binding affinity to HIV gp41.

Embodiments of the present invention include synthetic genes, whichencode novel antibody constructs of the present invention. Embodimentsof the present invention include monoclonal antibodies to HIV having atleast one dual variable domain chain with the dual domains linked by thelinker. Embodiments of the present invention may be derived frommonoclonal antibody 7B2.

Embodiments of the present invention provide methods of HIV treatmentand neutralization. HIV virus may be neutralized and/or eliminated withnovel constructs of the present invention. Novel constructs of thepresent invention may be administered for neutralization of HIV loadsand/or may include a cytotoxic agent to trigger cytotoxic activities ininfected cells. Embodiments of the present invention provide therapeutictreatments for HIV infections using novel DVD-Ig constructs. Embodimentsof the present invention provide methods of preventing HIV infection byadministering novel antibody constructs. The novel constructs of thepresent invention may be administered as part of a pharmaceuticalcomposition or as genetic constructs to be made into antibodies by thepatient's own cells.

An embodiment of the present invention includes an antibody having aheavy chain of SEQ ID NO 3 and a light chain of SEQ ID NO 5—construct#2816. An embodiment of the present invention includes an antibodyhaving a heavy chain of SEQ ID NO 4 and a light chain of SEQ ID NO6—construct #2817. An embodiment of the present invention includes anantibody having a heavy chain of SEQ ID NO 8 and a light chain of SEQ IDNO 5—construct #2858. An embodiment of the present invention includes anantibody having a heavy chain of SEQ ID NO 3 and a light chain of SEQ IDNO 7—construct #2859. An embodiment of the present invention includes anantibody having a heavy chain of SEQ ID NO 8 and a light chain of SEQ IDNO 6—construct #2860. An embodiment of the present invention includes anantibody having a heavy chain of SEQ ID NO 4 and a light chain of SEQ IDNO 7—construct #2861.

It is understood that upon binding of gp120 to CD4, the HIV Env proteinon the surface of the virion undergoes a conformational change thatexposes several key epitopes on both gp120 and gp41 that are vulnerableto attack by our immune systems. Substantial work has been published onthe potent neutralization activity of CD4 binding site (CD4bs)antibodies like VRC01 and b12. This class of antibodies binds to the CD4binding site on gp120 to block interaction. In addition to blocking theability of the virus to recognize its cognate receptors, theconformation changes expose areas of gp41 necessary for membrane fusionand cell entry.

It has been known that addition of soluble CD4 (sCD4) enhancesimmunoconjugate activity by changing the conformation of the Envprotein. Recently more CD4-inducible (CD4i) antibodies have beencharacterized; however, effective neutralization requires the presenceof CD4. This led to the problem of how to deliver both a soluble form ofCD4 and the monoclonal antibody simultaneously, which was solved byfusing the relevant portion of CD4 to a CD4i-antibody through a varietyof linkers. Several CD4i reagents, such as 17b, E51, and m9, have beenfused to sCD4 for enhanced activity. A range of CD4-i reagents have beenproduced from large, bispecific antibodies constructs to smallersingle-chain immunoadhesins. While the smaller scFv forms may result inhigher tissue penetration and epitope access, they are naturally lessavid and limited in their ability to stimulate the immune system throughFc interactions and antibody dependent cell cytotoxicity (ADCC). In thecase of CD4-7B2 constructs, the CD4 subunits are not only advantageousfor increasing neutralization but enhance exposure of the preciseepitope 7B2 requires.

Contemporary fusion antibodies were included in studies alongside apanel of CD4-7B2 DVD-Igs embodiments of the present invention. Thisallowed for a comparison of the effects of CD4 fusion versus mixingCD4i-antibodies with CD4-IgG. It would be much more efficient to supplya single engineered form of CD4-7B2, especially if it neutralizes anextremely broad range of HIV isolates. In embodiments of the presentinvention, CD4-7B2 IgGs were engineered to function as both animmunoconjugate and a neutralizing antibody. Results show that fusion ofCD4 to a single chain of 7B2 through a linker, such as a flexible linkeror a helical linker, creates an optimal configuration for binding theEnv subunits, neutralizing the infectivity of the virus, and killingcells already infected and producing the virus.

In addition to neutralization, immunoconjugates (IC) may be beneficialin reducing infectivity. HAART has contributed to a substantial decreasein viral loads, yet there is no vaccine or cure available. A potentialapproach would be use of an IC to eliminate the latent reservoir throughan “activate and purge” strategy advocated by inducing reactivation ofintegrated viral genomes and subsequent killing of infected cells. An ICis a chimeric protein that combines the targeting specificity of anantibody with the cellular effects of a toxin. Correctly chosen parentalantibodies can be incorporated into ICs which bind viral proteins on thesurface of infected cells where they are subsequently phagocytized,intracellularly processed, and then activated to kill the infected cell.Certain toxic molecules can induce apoptosis over necrosis which helpsreduce spread of virus to neighboring healthy cells. In this protocol,an HIV-activating agent would be administered first, and then treatmentwith ICs would deliver apoptosis-inducing drugs into infected cells.Early attempts have proved the concept that ICs can effectively killinfected cells in vitro using a CD4bs antibody and a bacterial exotoxin.In example embodiments of the present invention, ricin A chain (RAC) wasutilized due to its cytotoxic attributes and relative stability overtime. Extensive studies have shown how both native ricin and conjugatedricin are trafficked and activated to kill cells in vitro and in vivo.The new CD4-7B2 constructs of the present invention represent potentialtherapeutics with an increased breadth of neutralization and theunparalleled cell-targeted killing ability of ICs.

In an embodiment of the invention, the variable domains of thebispecific antibody disclosed herein comprise domains from antibodiesthat may bind the CD4 binding site, variable loops, and/or glycans ofgp120, and the variable domains from the anti-gp41 monoclonal antibody7B2, or other gp41 antibodies. The CD4-binding site of gp120 may betargeted with either CD4-itself (for example, soluble CD4) or anantibody or fragment thereof specific for this binding site. Suchantibody or fragment thereof may be monoclonal in nature. In embodimentsof the present invention, the variable domains may be linked by a linker(also known as an inter-V chain linker). Notably, unique construction ofthe inter-V chain linker may comprise synthetic peptides with eitherunordered or helical conformations.

Unlike conventional double-domain antibodies, which are symmetricassemblies of two identical heavy chains and two identical light chains,each containing identical variable regions, embodiments of the presentinvention may comprise asymmetric double variable domain antibodies,wherein the variable regions are not identical. For example, in oneembodiment, a heavy chain may contain a V_(H1)-L-V_(H2), and the lightchain variable region may consist of V_(L2) only. It should be notedthat any combination thereof may be used to make the asymmetricdouble-variable domain antibodies, such as antibodies that may bindgp120 and gp41. In embodiments of the present invention, symmetric orasymmetric heavy and light chains may be used.

It is generally known that tetrameric, symmetrical CD4-IgG neutralizesbetter than other CD4-IgG constructs, suggesting that symmetricalCD4-anti-gp41 antibodies may be effective neutralization antibodies.However, the neutralization capabilities of the symmetricalCD4-anti-gp41 antibodies were poor when experimentally tested,suggesting that CD4-anti-gp41 antibodies or antibody-like molecules maybe poor candidates for HIV-neutralization antibodies. Surprisingly, whenasymmetric CD4-anti-gp41 antibodies were constructed and tested inTZM-b1 cells against a panel of tier 1 and tier 2 clade B and C viruses,such antibodies had astonishingly high neutralization capacity. Thesesurprising results suggest that the asymmetric structure of theseantibodies may provide increased neutralization capacity.

An advantage of the disclosed invention is that the antibodies work wellas neutralizing antibodies. This is a unique advantage to theseantibodies, as other constructs were not as effective as neutralizingantibodies. For example, preliminary tests performed using 7B2-CD4hybrid IgG molecules in TZM-b1 cells against a panel of tier 1 and tier2 clade B and C viruses demonstrate that these constructs may be highlyeffective neutralizing antibodies. The 7B2 antibody served as areference antibody in these tests, and the CH01-31 antibody served as apositive control. In particular, the results from this experimentdemonstrate that CD4-anti-gp41 hybrid proteins disclosed herein arepotent neutralizers.

Embodiments of the invention may be employed to treat or prevent HIV. Inone such embodiment, the antibodies may be used as immunoconjugates,conjugated to a second molecule. For example, the second molecule may bea toxin, a label, a radioisotope, a drug, or a chemical compound.

In other embodiments, the antibodies disclosed herein may be used asneutralizing antibodies, passively administered or given via genetherapies. Supporting these approaches, preliminary data suggests thatcertain constructs may be highly effective neutralizing antibodies.

Generally, embodiments of the present invention comprise double variabledomain (DVD) antibodies that may bind in a tetravalent fashion toantigens of Env, such as gp120 and gp41. Antibodies to gp41 and/or gp120of Env may provide important neutralization components necessary for aneffective HIV/AIDS therapeutic, as Env is the only HIV protein displayedfully intact on the surface of HIV-infected cells. Embodiments of thepresent invention may incorporate monoclonal antibody 7B2, which bindsto the external loop of gp41. In certain embodiments of the presentinvention, asymmetrical DVD antibodies are provided.

Embodiments of the present invention provide immunoglobulins (Igs)engineered to improve HIV neutralization and/or delivery of cytotoxicagent to infected cells. Example embodiments include CD4-Igs containingdomains 1 and 2 of human CD4 attached to either heavy, light, or bothchains of full-length human 7B2 antibody, an IgG1/κ anti-gp41 antibody.Embodiments may be engineered using helical linkers or other flexiblesequences. For example, a helix-creating sequence (SEQ ID NO 9) or acombination of helical and flexible sequences (SEQ ID NO 10) may beincorporated as linkers. Embodiments may be created by expressing H andL chains in different plasmids and co-transfecting the H and L chainsinto 293F cells, creating CD4/7B2 chimeric Igs with various CD4-antibodyconformations and linker usage. A description of example embodiments ofthe present invention is set forth in Table 1, where construct nos.2816, 2817, and 2858 to 2861 represent novel embodiments of the presentinvention.

TABLE 1 Description of Parental Abs and Chimeric Constructs Name H ChainL Chain Linker Specificity 7B2 7B2 (γ1) 7B2 (κ) — gp41 CD4-IgG2 CD4-IgG2CD4 (κ) — gp120 CD4/7B2-Ig CD4-2H2-7B2 CD4-2H2-7B2 2H2* gp41/gp120 #2816CD4/7B2-Ig CD4-H4-7B2 CD4-H4-7B2 H4* gp41/gp120 #2817 CD4/7B2-Ig 7B2CD4-2H2-7B2 2H2 gp41/gp120 #2858 CD4/7B2-Ig CD4-2H2-7B2 7B2 2H2gp41/gp120 #2859 CD4/7B2-Ig 7B2 CD4-H4-7B2 H4 gp41/gp120 #2860CD4/7B2-Ig CD4-H4-7B2 7B2 H4 gp41/gp120 #2861 *2H2 is two flexibledomains flanking a helical core: [(GGGGS)

 (EAAAK)

 (GGGGS)

]

 H4 is the helical core only: [A-(EAAAK)

-A]

indicates data missing or illegible when filed

Full-length DVD-IgsCD4/7B2 chimeras with appropriately linked subunitswere produced in 293F cells in sufficient quantity and purity to testtheir ability to function as immunoconjugates, bind to infected cells,and neutralize HIV.

Binding of DVD-Igs to Recombinant and Native Antigen

ELISA was used to demonstrate binding of each construct to cognateantigens: gp41 peptide, recombinant gp160, or trimeric gp140. See, e.g.,FIG. 1. ELISA plates were coated with respective antigens, incubatedwith serial dilutions of Abs, and probed with an AP-conjugatedanti-human IgG secondary antibody. FIG. 1 has three graphs showing thebinding qualities of each construct and parental antibodies to gp41,gp160 and trimeric gp140. All CD4-Ig constructs bound each antigen inthe nanomolar range. As expected, CD4-IgG2 does not have any reactivitywith gp41, and binds the more “native” trimer gp140 better thannon-native gp160. 7B2, which identifies a linear epitope, binds well tothe peptide and gp160, but less well to trimeric gp140, suggesting thisepitope may be partially occluded in the trimer. There was no preferencefor antigen binding to gp41 based on attachment of CD4 to the light(#2860) or heavy chain (#2859, 2861), suggesting CD4 does not hinderaccessibility of 7B2 to its epitope. Studies with trimeric gp140 showedthat CD4/7B2 chimeras with CD4 on the heavy chain only (#2859, 2861)exhibited stronger binding than parental 7B2. And at the highest Igconcentration, #2861 exhibited the strongest binding to all antigenstested. Overall, the DVD-Igs perform as well or better than 7B2 andsCD4-IgG2, especially in the assays using the trimeric form of gp140,suggesting that recognition of the a more native conformation improvescumulative binding.

To test recognition of native Env by the chimeras, indirectimmunofluorescence and flow cytometry were used to analyze binding topersistently infected H9/NL4-3 cells. See, e.g., FIG. 2. In addition tothe novel constructs, which target both gp41 and gp120, binding of a setof gp120-specific CD4-Ig chimeras designed by others were also examined.It was found that binding of the CD4/Ig chimeras specific solely forgp120 exceeded binding of the constructs specific for both gp120 andgp41. Of the gp120/gp41 specific chimeras, construct #2817, with CD4fused to both heavy and light chains, bound best.

Immunoconjugate Cytoxicity

To determine whether the novel chimeras with CD4 linked to an anti-Envantibody can function as immunoconjugates to deliver cytotoxic agents toHIV-infected cells as a means to eradicate HIV infection, indirectimmunoconjugate assay was used to compare the constructs. H9/NL4-3 cellswere incubated with serial dilutions of Ab, then a ricin A chainconjugated to anti-IgG secondary Ab was added. Cell viability wasmeasured after 3 days. 7B2 was highly effective in targeting the toxinwhen sCD4 was present, but ineffective in its absence. See, e.g., FIG.3. CD4-Igs exhibited enhanced cytotoxicity compared to either parentalantibody alone and to all other CD4-linked constructs. BispecificCD4/7B2 targeting gp41 and gp120 exhibited more potent cytotoxicity thansimilar constructs only targeting gp120. No chimera was more effectivethan 7B2+sCD4. Among the CD4/7B2 constructs, those with two CD4 perconstruct (#2860 and #2861) outperformed the construct tetravalent withCD4 (#2817). The chimeras to the target cells and immunoconjugatekilling did not correlate each other. Those binding to gp120 aloneattached to the target cell better than those binding gp120/gp41, butkilled the same cells less well. Similarly the CD4/7B2 constructtetravalent for CD4 attached best, but was least affective fordelivering toxins.

HIV Neutralization

Although 7B2 has been shown to be an effective immunoconjugate, itcannot effectively neutralize most strains of HIV in conventional assaysof neutralization. On the other hand, CD4-IgG2 contains both the gp120binding site and the immunoglobulin constant region, and makes aneffective neutralizing antibody. To test the neutralization ability ofCD4/7B2-Ig constructs, both CXCR4 and CCR5-tropic HIV isolates were usedwith a TZM-b1 luciferase assay. Infectious virus stocks were premixedwith dilutions of antibodies, incubated with TZM-b1 cells for 3 days andthen luciferase activity was assayed. Constructs with CD4 linked to bothchains (#2817), the parental CD4-IgG2, and a panel of other CD4-linkedimmunoadhesins to four different HIV virus strains were tested first. Inall isolates tested, #2817 exhibited excellent neutralization activityand outperformed all other constructs. See, e.g., FIG. 4. In FACSexperiments with HIV-infected cells, anti-gp120 constructs showed strongbinding. Here they are able to neutralize several isolates at thehighest concentrations but quickly lose efficacy. VRC01 is the mosteffective for Ba-L and HT-594, while CD4/E51 neutralizes QZ4589 andHT-599. However, #2817 consistently displays enhanced neutralization ofall four strains across all concentrations. Other configurations ofCD4/7B2-Igs were tested against viral isolates ranging in difficulty toneutralize from easy (Ba-L) to hard (HT-92-594). FIG. 5 shows thatconstructs carrying CD4 on the heavy chain only (#2859 and 2861)neutralized all strains tested more efficiently than #2817 or any otherchimeras in the panel, even better than parental CD4-IgG2, which hasbeen used in phase II clinical trials. The most striking results wereobserved in the most difficult to neutralize strain, HT-92-594. TheCD4/7B2-Igs #2859 and #2861 neutralized at >1000× lower concentrationthan any other CD4 chimeric construct.

Further tests of neutralization were performed at the Duke UniversityHIV neutralization reference laboratories. Parental antibodies (7B2,CD4-IgG2), subunits (sCD4), and CD4/7B2-Igs (#2859, 2860, 2861), werecompared to a standard monoclonal antibody mixture (CH01-31).Neutralization of pseudovirus was tested in both TZM-b1 and A3R5.7 cellsusing tier 1 and 2 viruses from clades B or C. The CD4/7B2-Igs,especially #2861, showed broad and potent neutralizing activity acrossclades, consistently outperforming the parental antibodies, sCD4, or theCh01-31 standard. Constructs with CD4 fused to the heavy chain (#2859and 2861) outperformed the light chain fusion (#2860).

Materials and Methods

Reagents and Cells

H9 cells, human CD4+ lymphoma cell line, were obtained from Dr. M Reitz(Institute of Human Virology, Baltimore, Md.). H9/NL4-3 cells arepersistently infected with the NL4-3 molecular clone of HIV and retain aproductive infection in virtually 100% of tissue culture cells. TZM-b1cells (NIH AIDS Reagent Program, NIH-ARP) are HeLa cells expressing CD4,CCR5, and CXCR4, with a HIV-tat inducible luciferase andbeta-galactosidase reporter genes. H9/NL4-3 and TZM-b1 cells weremaintained at 37° in 5% CO₂ in RPMI 1640 medium with 10% fetal bovineserum (Gibco Invitrogen, Grand Island, N.Y.) as described elsewhere.

HIV isolates used in these studies were all Clade B, and include: NL4-3(X4-tropic), Ba-L (R5-tropic), 92HT594 (X4/R5), 92HT599 (X4), QZ4589(R5), and 96USHIPS7 (R5). All isolates were obtained from NIH-ARP andgrown in PHA blasts, with the exception of NL4-3, which was produced bythe H9/NL4-3 cell line.

Soluble, two-domain CD4 (sCD4; NIH-ARP) and CD4-IgG2 (PRO542; ProgeniesPharmaceuticals, Tarrytown, N.Y.) were used to observe CD4-mediatedeffects. Goat anti-human IgG (heavy+light chains) antibody wasconjugated to fluorescein isothiocyanate (FITC; Invitrogen) for flowcytometric analysis. Deglycosylated ricin A chain (RAC; obtained fromEllen Vitetta) was conjugated to purified anti-human IgG forcytotoxicity assays.

Design and Production of Antibodies

Synthetic genes encoding CD4/7B2-Ig constructs shown in table 1 weresynthesized, codon optimized for mammalian expression by GenScript(Piscataway, N.J.). Two additional mutations (T250Q and M428L) wereintroduced into the constant region of the heavy chain to increase invivo half-life of the antibody. DNA sequences were cloned into theeukaryotic expression plasmid pcDNA3.1 (Invitrogen) using eitherrestriction enzyme sites XbalI and PmeI for the heavy chain, or HindIIIand EcoRI for the light chain.

All antibodies were produced by transient transfection in suspension293F cells (Invitrogen, Carlsbad, Calif.) in serum-free Freestyleexpression media, shaking at 120 rpm in 8% Co_(e) at 37′ for transienttransfection. Synthetic Igs were purified by affinity chromatography onProtein A agarose beads (Invitrogen), and concentrated by MicroconYM-30k centrifugal filter (Millipore, Billerica, Mass.). All antibodyconcentrations were measured by bicinchoninic acid protein assay(Pierce, Rockford, Ill.) and confirmed using OD280. Microcapillaryelectrophoresis (Agilent Bioanalyzer, GE Healthcare) was used todetermine molecular weights and purity of products, and confirmconcentrations

Domains 1 and 2 of CD4 were joined to 7B2 using two effective linkersfrom previous studies: SEQ ID NO: 9 or SEQ ID NO: 10. The variabledomains of CD4 were fused to the N-terminus of either the 7B2 IgG1 heavychain, 7B2 kappa light chain, or both, creating a set of full lengthCD4/7B2-Ig.

ELISA

The antibodies tested, including the novel constructs, werecharacterized based on binding to Env (gp160) or its subunits (gp41,140) by indirect ELISA. The gp41 peptide has a linear sequence SEQ IDNO: 3 representing the epitope of 7B2. Gp160 antigen is a recombinantprotein consisting of the gp120 portion of MN and the gp41 portion ofLAI, designated MN/LAI (Quality Biological, Gaithersburg, Md.) andexpressed in mammalian cells. Gp140 is a trimeric version derived fromSF162 that was used to test binding to multimers. Immulon 2HB plates(Thermo, Walktham, Mass.) were coated with 1.0 ug/ml of antigen and theassay performed as described elsewhere, using AP-conjugated goatanti-human IgG (H+L chain specific) secondary antibody. ELISA plateswere read at 405 nm at room temperature in a BioTek EL320 microplatereader (BioTek, Winooski, Vt.) at 5-15 minute intervals. Time pointsshown in figures have been chosen so that maximal binding was within thedynamic range of the reader. Data are presented as the mean and SEM oftriplicate assays.

Indirect Immunofluorescence and Flow Cytometry

H9/NL4-3 cells (1×10̂5) were stained for flow cytometry in 100 ul inround bottom 96 well plates (Costar, Lowell, Mass.). Serial dilutions ofIg in PBA were added to the cells in the presence or absence of 500ng/ml sCD4. Cells were incubated 1 hr at room temperature, washed, thenstained with FITC-conjugated goat anti-human IgG (H+L chain specific)secondary antibody for 1-4 hrs, washed twice and fixed in 100 ul of 2%paraformaldehyde. After a minimum of 4 hrs, 150 ul PBS was added. Cellswere analyzed on a Becton-Dickinson LSR II (BD<Franklin Lakes, N.J.)with HTS plate reader. 10000 events were collected and data analyzed byFlo-Jo software (Treestar, Ashland, Oreg.). Forward scatter (FSC) andside scatter (SSC) gated data are represented as graphs of meanfluorescence. None of the parental or synthetic-Igs bound to uninfectedH9 cells.

Cytotoxicity Assay

An indirect cytotoxicity assay was performed to screen unconjugatedantibodies for their ability to kill infected cells. H9/NL4-3 cells(8×10̂3) were plated in triplicate. Controls included: no cells(background) and cells in the absence of antibody/IC (uninhibited).Serial dilutions of antibodies were incubated with cells for 1 hr in thepresence or absence of 300 μg/ml sCD4 in RPMI at 37°. The secondary ICwas affinity purified goat anti-human IgG (Invitrogen) conjugated todeglycosylated ricin A chain by the long chain heterobifunctional crosslinking reagent, succinimidyl6-[3(2-pyridyldithio)proprionamido]hexanoate (Pierce), using protocolsdescribed elsewhere [ ]. The secondary IC was added to a finalconcentration of 500 ng/ml. The plates were then incubated for 3 days.For the final 6 hrs of incubation, MTS/PMS substrate (Promega, Madison,Wis.) was added to each well and plates read hourly at 490 nm. Resultsrepresent the mean and SEM of triplicate samples, and are plotted as“Percent Killing” using the formula % kill=100*[(no Ab−Ab)/No Ab] withthe no cell background subtracted. Under these conditions, there was nocytotoxicity on uninfected H9 cells. To determine whether the ICactivity of the DVD-Ig represented an improvement over that of theparental antibodies, a one-tailed t-test comparing the DVD-Ig to themost effective parental antibody at each concentration was performed.

Direct cytotoxicity assay was performed with antibodies conjugated toricin A chain by the long chain heterobifunctional cross linkingreagent, succinimidyl 6-[3(2-pyridyldithio)proprionamido]hexanoate(Pierce. H9/NL4-3 cells (8×10̂3) were plated in triplicate in cRPMI in 96well flat-bottom tissue culture plates (Costar). Control included: nocells (background) and cells in the absence of IC, and cells +/−500μg/ml CD4-IgG2. Serial dilutions of ICs were incubated with cells for 3days in RPMI at 37°. For the final 6 hrs of incubation, MTS/PMSsubstrate (Promega) was added to each well and plates read hourly at 490nm. Results plotted similarly to indirect IC assay explained above.

Neutralization Assay

Neutralization of infectious HIV was measured in TZM-b1 cells, using aluciferase-read out assay. Each antibody was assayed in triplicate.Experiments included: background controls (cells, no virus, no antibody)and infected cells in the presence or absence of antibody. TZM-b1 cells(4×10̂4 cells/ml) were plated in 96-well plates with black sides andclear, flat bottom wells (Costar) and incubated overnight at 37° toallow attachment. The following day, 50 μl of serially dilutedantibodies in RPMI were mixed with 50 μl of a pretitered concentrationof virus and incubated for 1 hr at room temperature, then added to thecells in the presence of diethylaminoethyl dextran (Sigma) 15 μg/ml, andincubated for 6 hrs at 37°. Medium was added to a total volume of 200ul/well and plates incubated for 48 hr at 37°. For luciferase assays,medium was aspirated and 50 μl of Bright-Glo Lysis buffer (Promega) wasadded. Samples were frozen and thawed once, and incubated for 6 hr atroom temperature with orbital shaking at 120 rpm. Then 10 ul ofBright-Glo luciferase substrate (Promega) was added and luminescenceread on Bio-Tek KC4 plate reader as relative luminescence units. Resultsare displayed as percent neutralization (virus/no Ab=0%; no virus=100%neutralization) according to the formula:[1−(RLUAb−RLUbkgrd)/(RLUnoAb−RLUbackground)]*100.

Neutralization of pseudo-typed reference strains in TZM-b1 and A3R5.7cells was performed at the Duke University HIV neutralization referencelaboratories, using established assays.

Antibody-Dependent Cellular Viral Inhibition (ADCVI)

Embodiments of the present invention were tested to determine theirability to mediate ADCVI using human peripheral blood mononuclear cells(PBMC) as effector cells and CCR5+ CEM-NKr cells (AIDS Research andReference Reagent Program) infected 2 days earlier with HIVBAL at an moiof 0.02 as target cells. See, e.g., FIG. 6. Briefly, target cells werewashed twice in medium, then placed in wells of a V bottom plate at 104cells per well. Target cells were incubated in triplicate with mediumalone or diluted antibodies for 1 h at 37° C. and 5% CO₂. Freshlyisolated PBMC (105 per well) were then added. Four days later, thecultures were split ¼. On day 7, the medium in the wells was harvested,lysed with TritonX-100 detergent and analyzed for p24 content by ELISA.The % inhibition of infection was calculated after dividing the p24concentration in antibody cultures by the average p24 concentration incontrol cultures containing effector and target cells alone. Constructno. 2861 showed greater efficacy than either of the parental antibodies,or a mixture of both. See, e.g., FIG. 6.

Antibody-Dependent Phagocytosis (ADP)

Assays were performed using the THP-1 monocyte cell line and HIVgp140-coated fluorescent beads to evaluate ADP, specifically forconstruct no. 2861. See, e.g., FIG. 7. Briefly, 1.8×106neutravidin-coated 1 μm Fluorospheres (Invitrogen) were treated withrabbit anti-His tag antibody (Pierce), then washed and reacted withrecombinant gp140 SF162 protein (Immune Technology). After washingunbound material off the beads, they were incubated at 37° C. for 1 hwith dilutions of antibody in triplicate wells of a V-bottom plate.THP-1 cells (2×104 per well) were then added and incubated at 37° C. in5% CO2. After 4 h, the cells were washed with Ca+2/Mg+2-free DPBS andincubated at 37° C. for 10 min with 50 μl of 0.05% Trypsin/EDTA (LifeTechnologies). Cells were washed 2× in DPBS, re-suspended in 1%paraformaldehyde, then examined by flow cytometry for fluorescence. Thephagocytic score was calculated by multiplying the number ofbead-positive cells by the median fluorescent intensity. The averagescore obtained for triplicate wells of THP-1 and gp140-coated beadsincubated in medium alone was subtracted from all other scores prior tocalculating the average score for test samples. The ability of constructno. 2861 to promote phagocytosis of HIV-Env is greater than that of thecombination of the two parental Abs. See, e.g., FIG. 7.

While the embodiments are described with reference to variousimplementations and exploitations, it will be understood that theseembodiments are illustrative and that the scope of the invention is notlimited to them. Many variations, modifications, additions, andimprovements are also possible. Support for the present invention may befound in the attached documents and figures, all of which are expresslyincorporated herein in their entirety by reference hereto.

Sequence Listing SEQ ID NO 1 Length: 345 Type: amino acidOrganism: Human Immunodeficiency Virus Other Information: gp 41 of Env        10         20         30         40         50         60         70         |          |          |          |          |          |         |  AVGIGALFLG FLGAAGSTMG AASMTLTVQA RQLLSGIVQQ QNNLLRAIEA QQHLLQLTVW GIKQLQARIL        80         90        100        110        120        130        140         |          |          |          |          |          |         |  AVERYLKDQQ LLGIWGCSGK LICTTAVPWN ASWSNKSLEQ IWNHTTWMEW DREINNYTSL IHSLIEESQN       150         160       170        180        190        200        210         |          |          |          |          |          |         |  QQEKNEQELL ELDKWASLWN WFNITNWLWY IKLFIMIVGG LVGLRIVFAV LSIVNRVRQG YSPLSFQTHL       220        230        240        250        260        270        280         |          |          |          |          |          |         |  PTPRGPDRPE GIEEEGGERD RDRSIRLVNG SLALIWDDLR SLCLFSYHRL RDLLLIVTRI VELLGRRGWE       290        300        310        320        330        340         |          |          |          |          |          |ALKYWWNLLQ YWSQELKNSA VSLLNATAIA VAEGTDRVIE VVQGACRAIR HIPRRIRQGL ERILLSEQ ID NO 2 Length: 481 Type: amino acidOrganism: Human Immunodeficiency Virus Other Information: gp 120 of Env        10         20         30         40         50         60         70         |          |          |          |          |          |         |  TEKLWVTVYY GVPVWKEATT TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLVNVT ENFNMWKNDM        80         90        100        110        120        130        140         |          |          |          |          |          |         |  VEQMHEDIIS LWDQSLKPCV KLTPLCVSLK CTDLKNDTNT NSSSGRMIME KGEIKNCSFN ISTSIRGKVQ       150         160       170        180        190        200        210         |          |          |          |          |          |         |  KEYAFFYKLD IIPIDNDTTS YKLTSCNTSV ITQACPKVSF EPIPIHYCAP AGFAILKCNN KTFNGTGPCT       220        230        240        250        260        270        280         |          |          |          |          |          |         |  NVSTVQCTHG IRPVVSTQLL LNGSLAEEEV VIRSVNFTDN AKTIIVQLNT SVEINCTRPN NNTRKRIRIQ       290        300        310        320        330        340        350         |          |          |          |          |          |         |RGPGRAFVTI GKIGNMRQAH CNISRAKWNN TLKQIASKLR EQFGNNKTII FKQSSGGDPE IVTHSFNCGG       360        370        380        390        400        410        420         |          |          |          |          |          |         |EFFYCNSTQL FNSTWFNSTW STEGSNNTEG SDTITLPCRI KQIINMWQKV GKAMYAPPIS GQIRCSSNIT       430        440        450        460        470        480         |          |          |          |          |          |GLLLTRDGGN SNNESEIFRP GGGDMRDNWR SELYKYKVVK IEPLGVAPTK AKRRVVQREKSEQ ID NO 3 Length: 710 Type: amino acidOther Information: CD4-[2-helix-2]-7B2 DVR Heavy Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT        80         90        100        110        120        130        140         |          |          |          |          |          |         |KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT       150         160       170        180        190        200        210         |          |          |          |          |          |         |LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASGG       220        230        240        250        260        270        280         |          |          |          |          |          |         |GGSGGGGSLE AEAAAKEAAA KEAAAKEAAA KALEGGGGSG GGGSQVQLVQ SGGGVFKPGG SLRLSCEASG       290        300        310        320        330        340        350         |          |          |          |          |          |         |FTFTEYYMTW VRQAPGKGLE WLAYISKNGE YSKYSPSSNG RFTISRDANK NSVFLQLDRL SADDTAVYYC       360        370        380        390        400        410        420         |          |          |          |          |          |         |ARADGLTYFS ELLQYIFDLW GQGARVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS       430        440        450        460        470        480        490         |          |          |          |          |          |         |WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP       500        510        520        530        540        550        560         |          |          |          |          |          |         |PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN       570        580        590        600        610        620        630         |          |          |          |          |          |         |STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC       640        650        660        670        680        690        700         |          |          |          |          |          |         |LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT       710          | QKSLSLSPGK SEQ ID NO 4 Length: 690Type: amino acid Other Information: CD4-DVR Heavy Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |  MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT        80         90        100        110        120        130        140         |          |          |          |          |          |         |  KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT       150         160       170        180        190        200        210         |          |          |          |          |          |         |  LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASLE       220        230        240        250        260        270        280         |          |          |          |          |          |         |  AEAAAKEAAA KEAAAKEAAA KALEQVQLVQ SGGGVFKPGG SLRLSCEASG FTFTEYYMTW VRQAPGKGLE       290        300        310        320        330        340        350         |          |          |          |          |          |         |  WLAYISKNGE YSKYSPSSNG RFTISRDNAK NSVFLQLDRL SADDTAVYYC ARADGLTYFS ELLQYIFDLW       360        370        380        390        400        410        420         |          |          |          |          |          |         |  GQGARVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS       430        440        450        460        470        480        490         |          |          |          |          |          |         |  GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP       500        510        520        530        540        550        560         |          |          |          |          |          |         |  KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK       570        580        590        600        610        620        630         |          |          |          |          |          |         |  EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP       640        650        660        670        680        690                |          |          |          |          |          |        ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGKSEQ ID NO 5 Length: 502 Type: amino acidOther Information: CD4-[2-helix-2]-7B2 DVR Light Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |MTSTLPFSPQ VSTPRSKFKR ISSEFAATMN RGVPFRHLLL VLQLALLPAA TQGKKVVLGK KGDTVELTCT        80         90        100        110        120        130        140         |          |          |          |          |          |         |ASQKKSIQFH WKNSNQIKIL GNQGSFLTKG PSKLNDRADS RRSLWDQGNF PLIIKNLKIE DSDTYICEVE       150         160       170        180        190        200        210         |          |          |          |          |          |         |DQKEEVQLLV FGLTANSDTH LLQGQSLTLT LESPPGSSPS VQCRSPRGKN IQGGKTLSVS QLELQDSGTW       220        230        240        250        260        270        280         |          |          |          |          |          |         |TCTVLQNQKK VEFKIDIVVL AFQKASGGGG SGGGGSLEAE AAAKEAAAKE AAAKEAAAKA LEGGGGSGGG       290        300        310        320        330        340        350         |          |          |          |          |          |         |GSDIVMTQSP DSLAVSPGER ATIHCKSSQT LLYSSNNRHS IAWYQQRPGQ PPKLLLYWAS MRLSGVPDRF       360        370        380        390        400        410        420         |          |          |          |          |          |         |SGSGSGTDFT LTINNLQAED VAIYYCHQYS SHPPTFGHGT RVELRRTVAA PSVFIFPPSD EQLKSGTASV        430        440        450        460        470        480        490         |          |          |          |          |          |         |VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS       500          | SPVTKSFNRG EC SEQ ID NO 6 Length: 482Type: amino acid Other Information: CD4-DVR Light Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |  MTSTLPFSPQ VSTPRSKFKR ISSEFAATMN RGVPFRHLLL VLQLALLPAA TQGKKVVLGK KGDTVELTCT        80         90        100        110        120        130        140         |          |          |          |          |          |         |  ASQKKSIQFH WKNSNQIKIL GNQGSFLTKG PSKLNDRADS RRSLWDQGNF PLIIKNLKIE DSDTYICEVE       150         160       170        180        190        200        210         |          |          |          |          |          |         |  DQKEEVQLLV FGLTANSDTH LLQGQSLTLT LESPPGSSPS VQCRSPRGKN IQGGKTLSVS QLELQDSGTW       220        230        240        250        260        270        280         |          |          |          |          |          |         |  TCTVLQNQKK VEFKIDIVVL AFQKASLEAE AAAKEAAAKE AAAKEAAAKA LEDIVMTQSP DSLAVSPGER       290        300        310        320        330        340        350         |          |          |          |          |          |         |  ATIHCKSSQT LLYSSNNRHS IAWYQQRPGQ PPKLLLYWAS MRLSGVPDRF SGSGSGTDFT LTINNLQAED       360        370        380        390        400        410        420         |          |          |          |          |          |         |  VAIYYCHOYS SHPPTFGHGT RVELRRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN       430        440        450        460        470        480                 |          |          |          |          |          |        ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG ECSEQ ID NO 7 Length: 240 Type: nucleic acidOther Information: 7B2 Light Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |  METPAQLLFL LLLWLPDTTG DIVMTQSPDS LAVSPGERAT IHCKSSQTLL YSSNNRHSIA WYQQRPGQPP        80         90        100        110        120        130        140         |          |          |          |          |          |         |  KLLLYWASMR LSGVPDRFSG SGSGTDFTLT INNLQAEDVA IYYCHQYSSH PPTFGHGTRV ELRRTVAAPS       150         160       170        180        190        200        210         |          |          |          |          |          |         |  VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA       220        230        240          |          |          |DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC SEQ ID NO 8 Length: 475Type: amino acid Other Information: Parent 7B2 Heavy Chain        10         20         30         40         50         60         70         |          |          |          |          |          |         |MDWTWRVLFL VAAATGAHSQ VQLVQSGGGV FKPGGSLRLS CEASGFTFTE YYMTWVRQAP GKGLEWLAYI        80         90        100        110        120        130        140         |          |          |          |          |          |         |SKNGEYSKYS PSSNGRFTIS RDNAKNSVFL QLDRLSADDT AVYYCARADG LTYFSELLQY IFDLWGQGAR       150         160       170        180        190        200        210         |          |          |          |          |          |         |VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL       220        230        240        250        260        270        280         |          |          |          |          |          |         |SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KRVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM       290        300        310        320        330        340        350         |          |          |          |          |          |         |ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK        360        370        380        390        400        410        420         |          |          |          |          |          |         |VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK       430        440        450        460        470                      |          |          |          |          |             TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO 9Length: 22 Type: amino acid Other Information: helical linker        10         20          |          | LEAEAAAKEA AAKEAAAKEA AAKALESEQ ID NO 10 Length: 46 Type: amino acidOther Information: 2-Helix-2 linker        10         20          30         40          |          |          |          |GGGGSGGGGS LEAEAAAKEA AAKEAAAKEAI AAKALEGGGG SGGGGS

1-19 (canceled)
 20. An asymmetric double variable domain (DVD) antibody comprising at least one immunoglobulin heavy chain-light chain cross-linked pair, wherein the immunoglobulin heavy chain-light chain pair comprises a first variable domain having binding affinity for an epitope of an HIV gp41 polypeptide and further comprising a second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide, wherein the second variable domain is linked by a linker peptide to the first variable domain of the immunoglobulin heavy chain or immunoglobulin light chain, but is not linked to the first variable domains of both the immunoglobulin heavy chain and the immunoglobulin light chain.
 21. The asymmetric DVD-antibody of claim 20, wherein the second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide is a CD4 polypeptide or a fragment thereof.
 22. The asymmetric DVD-antibody of claim 20, wherein the first peptide domain having binding affinity for an epitope of an HIV gp41 polypeptide is the immunoglobulin variable region of monoclonal antibody 7B2.
 23. The asymmetric DVD-antibody of claim 20, wherein, in a heavy chain-light chain pair, the heavy chain comprises a first variable domain linked to a second variable domain and the light chain comprises a first variable domain but not a second variable domain.
 24. The asymmetric DVD-antibody of claim 20, wherein in a heavy chain-light chain pair, the light chain comprises a first variable domain linked to a second variable domain and the heavy chain comprises a first variable domain but not a second variable domain.
 25. The asymmetric DVD-antibody of claim 20, wherein the linker peptide comprises a helical core.
 26. The asymmetric DVD-antibody of claim 20, wherein the linker peptide comprises a helical core having a flexible domain at both the N-terminus and the C-terminus thereof.
 27. The asymmetric DVD-antibody of claim 20, wherein the heavy chain has an amino acid sequence selected from the group consisting of: SEQ ID Nos: 3, 4, and 8, and wherein the light chain has an amino acid sequence selected from the group consisting of: SEQ ID Nos: 5, 6, and 7, and wherein when the light chain has an amino acid sequence according to SEQ ID NO: 7, the heavy chain amino acid sequence is not according to SEQ ID NO:
 8. 28. The asymmetric DVD-antibody of claim 20, wherein the cross-linked heavy and light chains have cross-linked amino acid sequences selected from the group consisting of: SEQ ID Nos: 3 and 5, 4 and 6, 8 and 5, 3 and 7, 8 and 6, and 4 and
 7. 29. The asymmetric DVD-antibody of claim 20, wherein the cross-linked heavy and light chains have amino acid sequences selected from the group consisting of: SEQ ID Nos: 8 and 5, 3 and 7, 8 and 6, and 4 and
 7. 30. The asymmetric DVD-antibody of claim 20, wherein the linker peptide has an amino acid sequence according to SEQ ID NO:
 9. 31. The asymmetric DVD-antibody of claim 20, wherein the linker peptide has an amino acid sequence according to SEQ ID NO:
 10. 32. The asymmetric DVD-antibody of claim 20, wherein the artificial immunoglobulin is admixed with a pharmaceutically acceptable carrier.
 33. The asymmetric DVD-antibody of claim 20, wherein each of the heavy and the light chains is an expression product from an expression vector.
 34. The asymmetric DVD-antibody of claim 33, wherein at least one of the heavy and the light chain expression products from the expression vector has an N-terminal leader sequence attached thereto.
 35. A method of reducing the infectivity of an HIV by contacting said virus with an asymmetric DVD-antibody according to any of claims 20-34.
 36. A method of reducing the viability of an HIV-infected cell by the steps of: contacting a cell infected with a strain of HIV with an asymmetric DVD-antibody according to any of claims 21-35, whereby said DVD-antibody binds to a cell surface polypeptide of the infected cell forming a cell-DVD-antibody complex; and contacting said cell-DVD-antibody complex with a cytoxic agent capable of specifically recognizing the cell-bound DVD-antibody, thereby delivering the cytotoxic agent to the HIV-infected cell and reducing the viability of an HIV-infected cell.
 37. The method of claim 36, wherein the cytotoxic agent is an engineered anti-immunoglobulin IgG antibody conjugated to a cytotoxic polypeptide, wherein the engineered anti-immunoglobulin IgG antibody specifically binds to a constant region of the asymmetric DVD-antibody.
 38. The method of claim 36, wherein the cytotoxic polypeptide is a ricin A chain. 